UCSD-TV: Molecules for the Media

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Study Guide

What is it that those tiny bacterial invaders do that can bring down an organism so many times their size, and what can be done about it? This unit focuses on the molecular mechanisms of diseases caused by bacteria, but many of the strategies and tools used by scientists studying bacterial diseases are useful in the quest to understand any disease.

Molecular Medicine Takes on Microbe Invaders

Whether standing in a lush green forest, looking through a microscope at a drop of water, or walking along city streets, most organisms that we see: plants; animals; aquatic microbes called protists; and fungi are eukaryotes. Eukaryotes contain within their cells a nucleus and other organelles that are surrounded by membranes. In nearly every environment, both outside and inside the bodies of eukaryotes, are infinite numbers of tiny organisms: unicellular prokaryotes, non-cellular viruses and others. The prokaryotes, which include bacteria, do not have membrane bound organelles (no nucleus, etc.). Prokaryotes also usually have chemically complex cell walls. Features such as these that are unique to bacteria are of interest because they can be applied to designing medicines that treat bacterial infections without harming the host organism. Briefly review the differences between eukaryotes and prokaryotes .

A number of common antibiotics target a unique component of the cell walls of bacteria. It is the reason those antibiotics kill bacteria and not you; you do not have cell walls. Although some eukaryotes such as plants have them, their cell walls do not have a large molecule called peptidoglycan, which is present in many bacteria. Because it is unique to bacteria, it makes an ideal drug target. The building blocks of peptidoglycan are sugars (which are also the building blocks of complex carbohydrates or polysaccharides) and amino acids (which are also the building blocks of proteins).

Here is a flat diagram of how the peptidoglycan structure that makes up the cell wall of bacteria is situated outside the lipid bilayer, the plasma (cell) membrane. Remember that these layers are actually covering the entire outside of three-dimensional bacteria cells. Notice the connections between the peptidoglycan molecules. Those are not formed in new cells when the bacteria are dosed with antibiotics.

Image credit: Ann Marie Wellhouse

Many of the chemicals that are used as antibiotics against bacteria keep the peptidoglycan structure from forming by disrupting the enzymes (proteins) that catalyze the chemical reactions to build the structure. The cell walls of bacteria dosed with antibiotics do not become rigid because the peptidoglycan structure is what makes them inflexible. If the cell wall is loose, then the bacterium is not able to hold all of the cell chemicals in and the cell ruptures into a gooey mass that is quickly gobbled up by the immune system. Find out more about peptidoglycan and how it is made.

Penicillin is an example of an antibiotic that targets the peptidoglycan chains in the cells of bacteria. Read a discussion of how penicillin kills bacteria. Also, watch an animation of how penicillin cures.

Like penicillin, the first antibiotic discovered, many antibiotics work by interfering with the formation of the bacterial cell wall. However, other antibiotics work via completely different mechanisms, for example by interfering with protein synthesis or the synthesis of RNA or DNA. see: View a list of different types of antibiotics and the mechanism by which they kill bacteria.

Although this unit is focused on disease-causing bacteria, it is important to keep in mind that we should not lump all bacteria together and think of them as public enemy number one. “We each carry two to five pounds of live bacteria in our bodies. Some, like Clostridium difficile, are potentially harmful. Many bacteria, however, are quite useful—so useful, in fact, that we could not live without them.” (From the Howard Hughes Medical Institute Bulletin)

How do Bacteria Develop Resistance?

Antibiotic resistance is a serious problem. Bacteria do not passively accept the drugs and die, they keep coming up with new ways to destroy them. Certain bacteria have developed resistance to multiple antibiotics that work via different mechanisms. It is why researchers are constantly trying to develop new antibiotics. In addition, bacteria can exchange genes that provide resistance to antibiotics. Bacteria transfer these genes to each other as plasmids—small, circular pieces of DNA—in a process called conjugation. Watch an animated slide show to learn more about the various processes by which bacteria can resist antibiotics.

Microbial Invasion!

Toxins produced by certain bacteria can be deadly. These toxins are proteins—virulence factors—that serve to support bacterial growth. Even when antibiotics keep the bacteria from reproducing, if the infection has gone on for a while before antibiotic treatment begins, the millions of bacteria already present may continue to produce lots of toxin. Those toxins may severely damage or kill the host. For instance, particularly lethal strains of Pseudomonas aeruginosa bacteria inject a toxin called ExoU into mammalian cells. Partho Ghosh, professor of chemistry and biochemistry at UCSD, and colleagues have discovered that the toxin destroys mammalian cells by degrading their cell membranes. You can learn more by watching the video presentation by Professor Ghosh, “Molecular interactions between hosts and pathogens.”

The image shows a mammalian cell with bacterial toxin, ExoU, (green spots because it is linked to a fluorescent protein) at the cell membrane. Credit: Rebecca Phillips, UCSD

The bacteria that cause anthrax also produce toxins. Read the following excerpt from a journal paper on anthrax by Thiang Yian Wong, Robert Schwarzenbacher, and Robert Liddington at the Burnham Institute in La Jolla, California:

“Anthrax Toxin, together with its bacterial capsule, is a major virulence factor in anthrax. The virulent strain of Bacillus anthracis is an encapsulated gram-positive, rod-shaped, spore-forming bacterium that produces and exports the three Anthrax Toxin proteins, Protective Antigen (PA), Lethal Factor (LF) and Edema Factor (EF). The infectious agent of anthrax, the bacillus spore, may be introduced into a mammalian host through inhalation, ingestion and subcutaneous (beneath the skin) wounds. Once in a susceptible host organism, the spores germinate within macrophages (white blood cells) and their vegetative cells proliferate rapidly. The explosive release of bacilli (new anthrax bacteria) from the host macrophages simultaneously introduces the three toxin components into the bloodstream.”
- (Credit)

Anthrax is one of very few bacteria that can tolerate drying out by forming spores. Learn more about how bacteria form spores.

Learn more about how the Anthrax toxin works.

What Do Enzymes Do?

All of the structures and functions of living organisms are the results of endless complex chemical reactions. Proteins play a major role in the biochemistry of living organisms. Everyone eats proteins. Some people count them or compare them to carbs. Lots of people eat candy bars that claim to supply them. Vegetarians prefer cheese and beans as a source of protein and carnivores and omnivores prefer chewing on other creatures. Proteins are made up of hundreds or thousands of smaller units called amino acids.

The plasma membrane has many embedded proteins that serve diverse purposes. Image credit: Ann Marie Wellhouse

Proteins that are catalysts are called enzymes. Enzymes facilitate chemical reactions—that is they make reactions happen by bringing molecules together and providing a chemical environment, within the enzyme, that helps the molecules react. Enzymes are not used up in the process. There are many different kinds of enzymes; for example, an enzyme that removes a phosphate group from ATP to put it on another molecule is called a kinase. An enzyme that breaks down other proteins is a protease.

Enzymes have an active site (dark blue) where they hook onto the molecule(s) that will react—the substrate (purple). After the reaction takes place, new products are formed. The following illustrates a protein being broken down by a protease.

Image credit: Ann Marie Wellhouse

The enzyme active site and the substrate fit together specifically in shape and charge. When researchers want to find a way to block the action of a bacterial enzyme that can damage the host, they look for a chemical that can fit in the active site the same way, to block the enzyme from binding with the normal substrate.

Seth Cohen, a professor of chemistry and biochemistry at UCSD, and colleagues are currently studying how certain chemicals can bind directly to a metal ion, such as zinc, that is in the active site of some bacterial enzymes. They are also interested in metal-containing enzymes involved in diseases, such as arthritis, that are not caused by bacteria. The researchers are attempting to use the affinity for active site metals as a guidance system, so that the new medicines will only bind to that specific active site and not produce unwanted side effects. To learn more about this research, you can watch Professor Cohen’s video presentation, “The chink in the armor: taking aim at metals in enzymes.”

A protein consists of amino acids strung together, but proteins fold into an intricate three-dimensional structure. The shape of the protein depends on how the different amino acids are strung together. Some amino acids have chemical groups that are hydrophobic—water-hating—which tend to be contained within the folds of the protein, or embedded in cell membranes. Other amino acids have chemical groups that are hydrophilic—water-loving—and are usually in contact with the aqueous solution within or surrounding a cell. The protein plays a bit of molecular “Twister” permitting the different amino acids to be in their optimal environment. The twisted structure may be stabilized by amino acids that can link up with other amino acids via covalent bonds.

The protein’s three-dimensional structure is absolutely essential to its function. Therefore, drugs must be designed in a way that takes a protein’s three-dimensional structure into consideration. It is essential to develop tools and techniques that can be used to determine the three dimensional structure of a protein, and understand how proteins interact with each other and with a substrate. Elizabeth Komives, a professor of chemistry and biochemistry at UCSD, and her colleagues have developed a technique called MALDI-Mapping that makes it possible to determine how the structure of an enzyme changes when a substrate binds to it. For example, her group has used the technique to understand interactions between proteins involved in blood-clotting, and is currently using it to study proteins that play a role in Alzheimer’s disease. To learn more about this research, you can watch Professor Komives video presentation, “A biochemist’s tool kit to study the battlefront up close.”

Laboratory Activity: Microscopic Examination of Bacteria

Before beginning the laboratory activity, read about the differences between Gram-positive bacteria, such as anthrax, and Gram-negative bacteria such as E. coli.

Q. Are Gram-negative or Gram-positive bacteria resistant to penicillin? Why (hint: how does penicillin kill bacteria)?

Activity:

Note: Use gloves while performing this lab activity.

From your teacher, obtain a living bacterial culture of E. coli. E. coli are bacteria that normally live in the gut. They are Gram negative and rod-shaped.
Make a smear slide as follows:

Image credit: Ann Marie Wellhouse
Put the used cover slip in 10% bleach water to kill the bacteria before discarding.
Turn on a gas burner (a candle or hair drier that heats without blowing also work).
Hold the slide so that the flame can warm it but do not barbeque the bacteria! The idea is to dry the slide but leave the bacteria intact. If the slide becomes too hot to handle in your plastic-gloved hand the bacteria are fried. If that happens, make a new slide.
When your smear is dry, stain it using the Gram Staining Method.

Q. What color are the Gram-negative E. coli rods? Why?

Watch an animation on the different shapes of bacterial types.

	Study Guide \| Explore This Topic \| Meet the Scientist Links & Resources \| Teacher Resources \| Overview Study Guide What is it that those tiny bacterial invaders do that can bring down an organism so many times their size, and what can be done about it? This unit focuses on the molecular mechanisms of diseases caused by bacteria, but many of the strategies and tools used by scientists studying bacterial diseases are useful in the quest to understand any disease. Molecular Medicine Takes on Microbe Invaders Whether standing in a lush green forest, looking through a microscope at a drop of water, or walking along city streets, most organisms that we see: plants; animals; aquatic microbes called protists; and fungi are eukaryotes. Eukaryotes contain within their cells a nucleus and other organelles that are surrounded by membranes. In nearly every environment, both outside and inside the bodies of eukaryotes, are infinite numbers of tiny organisms: unicellular prokaryotes, non-cellular viruses and others. The prokaryotes, which include bacteria, do not have membrane bound organelles (no nucleus, etc.). Prokaryotes also usually have chemically complex cell walls. Features such as these that are unique to bacteria are of interest because they can be applied to designing medicines that treat bacterial infections without harming the host organism. Briefly review the differences between eukaryotes and prokaryotes . A number of common antibiotics target a unique component of the cell walls of bacteria. It is the reason those antibiotics kill bacteria and not you; you do not have cell walls. Although some eukaryotes such as plants have them, their cell walls do not have a large molecule called peptidoglycan, which is present in many bacteria. Because it is unique to bacteria, it makes an ideal drug target. The building blocks of peptidoglycan are sugars (which are also the building blocks of complex carbohydrates or polysaccharides) and amino acids (which are also the building blocks of proteins). Here is a flat diagram of how the peptidoglycan structure that makes up the cell wall of bacteria is situated outside the lipid bilayer, the plasma (cell) membrane. Remember that these layers are actually covering the entire outside of three-dimensional bacteria cells. Notice the connections between the peptidoglycan molecules. Those are not formed in new cells when the bacteria are dosed with antibiotics. Image credit: Ann Marie Wellhouse Many of the chemicals that are used as antibiotics against bacteria keep the peptidoglycan structure from forming by disrupting the enzymes (proteins) that catalyze the chemical reactions to build the structure. The cell walls of bacteria dosed with antibiotics do not become rigid because the peptidoglycan structure is what makes them inflexible. If the cell wall is loose, then the bacterium is not able to hold all of the cell chemicals in and the cell ruptures into a gooey mass that is quickly gobbled up by the immune system. Find out more about peptidoglycan and how it is made. Penicillin is an example of an antibiotic that targets the peptidoglycan chains in the cells of bacteria. Read a discussion of how penicillin kills bacteria. Also, watch an animation of how penicillin cures. Like penicillin, the first antibiotic discovered, many antibiotics work by interfering with the formation of the bacterial cell wall. However, other antibiotics work via completely different mechanisms, for example by interfering with protein synthesis or the synthesis of RNA or DNA. see: View a list of different types of antibiotics and the mechanism by which they kill bacteria. Although this unit is focused on disease-causing bacteria, it is important to keep in mind that we should not lump all bacteria together and think of them as public enemy number one. “We each carry two to five pounds of live bacteria in our bodies. Some, like Clostridium difficile, are potentially harmful. Many bacteria, however, are quite useful—so useful, in fact, that we could not live without them.” (From the Howard Hughes Medical Institute Bulletin) How do Bacteria Develop Resistance? Antibiotic resistance is a serious problem. Bacteria do not passively accept the drugs and die, they keep coming up with new ways to destroy them. Certain bacteria have developed resistance to multiple antibiotics that work via different mechanisms. It is why researchers are constantly trying to develop new antibiotics. In addition, bacteria can exchange genes that provide resistance to antibiotics. Bacteria transfer these genes to each other as plasmids—small, circular pieces of DNA—in a process called conjugation. Watch an animated slide show to learn more about the various processes by which bacteria can resist antibiotics. Microbial Invasion! Toxins produced by certain bacteria can be deadly. These toxins are proteins—virulence factors—that serve to support bacterial growth. Even when antibiotics keep the bacteria from reproducing, if the infection has gone on for a while before antibiotic treatment begins, the millions of bacteria already present may continue to produce lots of toxin. Those toxins may severely damage or kill the host. For instance, particularly lethal strains of Pseudomonas aeruginosa bacteria inject a toxin called ExoU into mammalian cells. Partho Ghosh, professor of chemistry and biochemistry at UCSD, and colleagues have discovered that the toxin destroys mammalian cells by degrading their cell membranes. You can learn more by watching the video presentation by Professor Ghosh, “Molecular interactions between hosts and pathogens.” The image shows a mammalian cell with bacterial toxin, ExoU, (green spots because it is linked to a fluorescent protein) at the cell membrane. Credit: Rebecca Phillips, UCSD The bacteria that cause anthrax also produce toxins. Read the following excerpt from a journal paper on anthrax by Thiang Yian Wong, Robert Schwarzenbacher, and Robert Liddington at the Burnham Institute in La Jolla, California: “Anthrax Toxin, together with its bacterial capsule, is a major virulence factor in anthrax. The virulent strain of Bacillus anthracis is an encapsulated gram-positive, rod-shaped, spore-forming bacterium that produces and exports the three Anthrax Toxin proteins, Protective Antigen (PA), Lethal Factor (LF) and Edema Factor (EF). The infectious agent of anthrax, the bacillus spore, may be introduced into a mammalian host through inhalation, ingestion and subcutaneous (beneath the skin) wounds. Once in a susceptible host organism, the spores germinate within macrophages (white blood cells) and their vegetative cells proliferate rapidly. The explosive release of bacilli (new anthrax bacteria) from the host macrophages simultaneously introduces the three toxin components into the bloodstream.” - (Credit) Anthrax is one of very few bacteria that can tolerate drying out by forming spores. Learn more about how bacteria form spores. Learn more about how the Anthrax toxin works. What Do Enzymes Do? All of the structures and functions of living organisms are the results of endless complex chemical reactions. Proteins play a major role in the biochemistry of living organisms. Everyone eats proteins. Some people count them or compare them to carbs. Lots of people eat candy bars that claim to supply them. Vegetarians prefer cheese and beans as a source of protein and carnivores and omnivores prefer chewing on other creatures. Proteins are made up of hundreds or thousands of smaller units called amino acids. The plasma membrane has many embedded proteins that serve diverse purposes. Image credit: Ann Marie Wellhouse Proteins that are catalysts are called enzymes. Enzymes facilitate chemical reactions—that is they make reactions happen by bringing molecules together and providing a chemical environment, within the enzyme, that helps the molecules react. Enzymes are not used up in the process. There are many different kinds of enzymes; for example, an enzyme that removes a phosphate group from ATP to put it on another molecule is called a kinase. An enzyme that breaks down other proteins is a protease. Enzymes have an active site (dark blue) where they hook onto the molecule(s) that will react—the substrate (purple). After the reaction takes place, new products are formed. The following illustrates a protein being broken down by a protease. Image credit: Ann Marie Wellhouse The enzyme active site and the substrate fit together specifically in shape and charge. When researchers want to find a way to block the action of a bacterial enzyme that can damage the host, they look for a chemical that can fit in the active site the same way, to block the enzyme from binding with the normal substrate. Seth Cohen, a professor of chemistry and biochemistry at UCSD, and colleagues are currently studying how certain chemicals can bind directly to a metal ion, such as zinc, that is in the active site of some bacterial enzymes. They are also interested in metal-containing enzymes involved in diseases, such as arthritis, that are not caused by bacteria. The researchers are attempting to use the affinity for active site metals as a guidance system, so that the new medicines will only bind to that specific active site and not produce unwanted side effects. To learn more about this research, you can watch Professor Cohen’s video presentation, “The chink in the armor: taking aim at metals in enzymes.” A protein consists of amino acids strung together, but proteins fold into an intricate three-dimensional structure. The shape of the protein depends on how the different amino acids are strung together. Some amino acids have chemical groups that are hydrophobic—water-hating—which tend to be contained within the folds of the protein, or embedded in cell membranes. Other amino acids have chemical groups that are hydrophilic—water-loving—and are usually in contact with the aqueous solution within or surrounding a cell. The protein plays a bit of molecular “Twister” permitting the different amino acids to be in their optimal environment. The twisted structure may be stabilized by amino acids that can link up with other amino acids via covalent bonds. The protein’s three-dimensional structure is absolutely essential to its function. Therefore, drugs must be designed in a way that takes a protein’s three-dimensional structure into consideration. It is essential to develop tools and techniques that can be used to determine the three dimensional structure of a protein, and understand how proteins interact with each other and with a substrate. Elizabeth Komives, a professor of chemistry and biochemistry at UCSD, and her colleagues have developed a technique called MALDI-Mapping that makes it possible to determine how the structure of an enzyme changes when a substrate binds to it. For example, her group has used the technique to understand interactions between proteins involved in blood-clotting, and is currently using it to study proteins that play a role in Alzheimer’s disease. To learn more about this research, you can watch Professor Komives video presentation, “A biochemist’s tool kit to study the battlefront up close.” Laboratory Activity: Microscopic Examination of Bacteria Before beginning the laboratory activity, read about the differences between Gram-positive bacteria, such as anthrax, and Gram-negative bacteria such as E. coli. Q. Are Gram-negative or Gram-positive bacteria resistant to penicillin? Why (hint: how does penicillin kill bacteria)? Activity: Note: Use gloves while performing this lab activity. From your teacher, obtain a living bacterial culture of E. coli. E. coli are bacteria that normally live in the gut. They are Gram negative and rod-shaped. Make a smear slide as follows: Image credit: Ann Marie Wellhouse Put the used cover slip in 10% bleach water to kill the bacteria before discarding. Turn on a gas burner (a candle or hair drier that heats without blowing also work). Hold the slide so that the flame can warm it but do not barbeque the bacteria! The idea is to dry the slide but leave the bacteria intact. If the slide becomes too hot to handle in your plastic-gloved hand the bacteria are fried. If that happens, make a new slide. When your smear is dry, stain it using the Gram Staining Method. Q. What color are the Gram-negative E. coli* rods? Why?* Watch an animation on the different shapes of bacterial types. ©2010 Regents of the University of California. Terms and Conditions of Use.		Watch now in Flash Player (English; 56 minutes) For specific clips, scroll ahead to certain segments in RealPlayer. For example, if the segment is (8m:45s -- 22m:54s), then the clip begins at 8 minutes and 45 seconds and ends at 22 minutes and 54 seconds. Introduction (0m:0s -- 8m:45s) The chink in the armor: taking aim at metals in enzymes (8m:45s -- 22m:54s) Molecular interactions between hosts and pathogens (22m:54s -- 35m:33s) A biochemist's tool kit to study the battlefront up close (35m:33s -- 55m:03s)